UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA
DOCTORAL SCHOOL FACULTY OF HORTICULTURE
Ing. Adriana-Daniela BACIU
SUMARY OF PhD THESIS Research regarding the genetical enrichment
for the Calendula
Scientific Supervisor: Prof. Dr. Radu SESTRAŞ
Cluj-Napoca 2011
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CONTENT Introduction….......………………………………………………………………........…….... 1 IMPORTANCE, USAGE AND CURENT RESEARCH STATUS REGARDING THE IMPROVEMENT OF CALENDULA GENDER………………………………………………
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Calendula economical and social importance………………………………………... 1 Main utility of Calendula gender plants……………………………………………... 1 Calendula usage as ornamental plant………………………………………… 1 Calendula usage as therapeutically plant…………………………………….. 2 Calendula usage as food……………………………………………………… 2 Plant breading importance at Calendula gender……………………………………... 2 Plant breeding methods for Calendula gender………………………………. 2 BIOLOGICAL MATERIAL AND WORK PROTOCOLS………………………………….. 3 Experimental plot layout……………………………………………………... 3 Appling of Griffing model II dialel breading method................................................... 4 Study of main seed characteristics at 46 Calendula genotypes………………………. 4 Study of main plants characteristics………………………………………………….. 4 Plant behaviour to pest and diseases attack…………………………………………... 4 Methods used for the determination of the philogenetic relationships……………….. 4 Molecular markers method............................................................................... 4 RAPD Random Amplified Polymorphic Method............................................. 5 Artificial breading system…………………………………………………………….. 5 Intraspecific and interspecific breading............................................................ 5 Artificial breading protocol………………………………………………….. 5 Experimental data processing........................................................................................ 5 Statistically analysis of variance……………………………………………... 5 Genetically analysis of variance……………………………………………... 5 Heritability coefficient calculation………………………………………....... 5 Statistical and mathematical methods used for calculation and interpretation of molecular variability study………...............................................................
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RESULTS AND DISCUSIONS………………………………………………………………. 6 Results regarding the variability of main seed characteristics at 46 Calendula genotypes……………………………………………………………………………..
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Results regarding the variability of main plant characteristics at 45 Calendula genotypes……………………………………………………………………………...
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Phenotypic correlation results for main seed and plant characteristics at Calendula…. 8 Results regarding the behavior of 45 Calendula genotypes at aphid attack…………... 9 Results regarding the philogenetic relationships between different species and genotypes………………………………………………………………………………
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Dendogram result regarding the 45 genotypes belonging to six species of Calendula……………………………………………………………………..
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Obtained results during studies conducted at F1 Calendula hybrids prevailed through intraspecific dialel breadings, Griffing II model……………………………………...
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Growth elements analyses at F1 hybrids…………………………………….. 13 Hibrid height…………………………...…………………………………….. 13 Heritability of analized characteristics at F1 intraspecific Calendula hybrids, obtained by Griffin II model breading…………………………………………..…….
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Conclusions and recommendations……………………….......................……………..…… 15Selective bibliography………………………….....…….…………...………..……...……… 17
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INTRODUCTION
The PhD thesis entitled „Research regarding the genetical enrichment at Calendula” aims to study phenotypic and molecular variability in different genotypes of the Calendula genus genitors identifying suitable targets for improvement and achievement of modern intra-and interspecific hybridization in order to create artificial variability necessary for creating new cultivars, and study the genetic determinism of the main issues of agronomic interest.
Research motivation for choosing the theme consisted in the desire to study further the species of the Calendula gender, especially the C. officinalis L. species and enrichment of the existing germplasm fund with new genotypes, in order to achieve a selection of useful bases for the creation of new varieties. Calendula gender belongs to the Asteraceae family composing over 25 species native in the Mediterranean and west Asia, Central Europe and South-East and North Africa (MEUSEL and OHLE, 1966; HEYN and all, 1974; www.nf-2001.org).
The research study distinguish its self by the biological material studied and work protocols upon phenotipical and molecular variability, heredity study for some main important characteristics on hybrid progeny executed for the first time in Romania. According to this results was conceived a database with phenotipical characteristics of plants and seeds, useful for further genotype observing and identifications. The final results can be useful in further works for obtaining new genotypes and parental lines for new breading processes. In the firs stage of the PhD thesis was elaborated a thorough study of national and international literature concerning the studied issue.
A phenotypic and molecular study was conducted upon 45 genotypes belonging to six species of Calendula. Parental identification for causing artificial variability and genetic determinism study was established through classical and modern methods. The molecular characterization was performed using RAPD markers; the final results estimated the degree of genetic relatedness between species visible by creating a specific dendogram.
IMPORTANCE, USAGE AND CURENT RESEARCH STATUS REGARDING THE IMPROVEMENT OF CALENDULA GENDER
CALENDULA ECONOMICAL AND SOCIAL IMPORTANCE
Marigolds are considered to be one of the most popular ornamental plants, largely spread
and with high ornamental values. Regarding economical aspects the importance of this culture has increased due to a broader range of use in raw materials such as flowers, leaves and seeds (JANISZOWSKA, 1985). During the last decade in the E.U. area where established „Implementation and research programs” of C. officinalis L. species (www.nf-2001.org).
MAIN UTILITY OF CALENDULA GENDER PLANTS
From approximately 25 species of Calendula gender, the mostly used species in production, human and veterinary medicine and different industries is C. officinalis L.
Calendula usage as ornamental plant: marigolds were first used as decorative plants
duet o their inflorescences, and later as medicinal plants, once with the discovery and appreciation of their healing qualities (KEMPER, 1999; GONCEARIUC, 2003). From ancient times Egyptians, Greeks, Hindus and Arabs cultivated these plants due to their beauty (VERZEA, 2001). From esthetical point of view are used genotypes with abundant flowers, colored in yellow, yellow-orange, orange, with high waist and multiple petals in inflorescence.
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Calendula usage as therapeutically plant: Dioscorides (40-90 d. Hristos) military doctor and Greek philosopher, described for the very first time in its treaty intitled „About medication” marigolds as medicinal plants. The paper was considered to be a medical knowledge synthesis, kindly appreciated by European medics (COIFAN, 2003).
- In human medicine: prepared as tea, infusion, decoctions, tincture (BLUMENTHAL, 1998), was efficient for liver illness, cramps, ulcers, jaundice, hemorrhoids, internal healing (GRAF, 2000), skin cancer treatment (MANOLOV, 1964). With a widely specter of action: choleretic, antispasmodic, bactericidal (MIDDLETON, 1996), anticancer (AMOSOVA and all., 1991), wounds, frostbite, burns, acne (BRUNETON, 1995). Applied as lotion: for skin illness, ulcerations, frostbite, burns, fungus (JORGE and all., 1996). Extracts derived from dried flowers inhibit the replication of HIV-1 (KALVATCHEV, 1997).
- In veterinary medicine: the raw material represented by marigolds is used for the preparation of different tinctures (GRELA, 1998). The tincture obtained from C. officinalis L. and C. arvensis L. has homeopathic and antiviral properties (BOGDANOVA, 1970), sedative and hypertensive effect (BOJADIEV, 1964). La USAMV Cluj-Napoca, have been conducted studies regarding the effect of alcoholic extract of C. officinalis L. upon cell immunity at equines (BOLFĂ, 2010). Calendula usage as food: as salads, condiment, replacement of saffron (ISMAGILOV, 2000) from ligulate flowers is obtaining alimentary colorants, used in margarine industry, butter industry (PĂUN, 1995). Leafs: for salad aromatization. Petals can also be used for salad aromatization, in sups, cookies used as condiments (MUSTEAŢĂ and BRÂNZILĂ, 1997).
PLANT BREADING IMPORTANCE AT CALENDULA GENDER Through plant breading techniques is intended to create valuable cultivars that will outcome existing cultivars. Plant breading enrolls „ all the methods used to obtain and maintain genetic creations based on specific methodology of investigation and enforcement” (SAVATTI and all., 2004). First ornamental plants breading techniques are older as their first time cultivation (ARDELEAN, 1986). In Romania outstanding accomplishments for ornamental plants breading outcome after the establishment of ICAR, in 1927 in specific laboratories of biology and breading (SESTRAŞ, 2004).
Plant breeding methods for Calendula gender Natural hybrids selection – enrolls the removal of unsuitable plant propagation and
breeding only those that prove adequate for the purpose intended by the breeder (ARDELEAN and all., 2006).
Intraspecific hybridization – prevailing is conditioned by a series of conditions: objective delimitation, existent source of genes and choosing the proper ones, to obtain a certain size of hybrid progenies (ARDELEAN, 1986).
During breeding processes was observed that the most efficient selection method is related to the size of anthodium and the number of chromosomes 2n=32. In many cases biotypes of marigold with large flowers have a genome of 2n=32 (LEWANDOWSKI and BRZKIN, 1982). In Romania was obtained Petrana variety due to repeated intraspecific breadings with a cehozlovach variety named Plamen and a local population from Albota (DIACONU, 1991). In Moldavian Republic: Nataly and Diana varieties by repeated intraspecific breadings (GONCEARIUC, 2003). For medicinal purposes the selection activity is keen on genotypes with ligulate flowers colored in dark-orange (CRĂCIUN, 1976).
Interspecific breading – consists as a method that lead to large variability, used in breading processes from the end of XIX century. Active compounds useful for medicine are contained in only two species from 25 existent: C. officinalis L. and C. arvensis L. (BRÂNZILĂ, 2005). For reaching these breading purposes we must bee keen on intraspecific breadings between C. officinalis L. and C. arvensis L. species.
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BIOLOGICAL MATERIAL AND WORK PROTOCOLS Table 1 presents 46 genotypes derived from 6 Calendula species prevailed from 12
countries all over the world. The biological material vas prevailed by seed exchange between Agrobotanical Garden of USAMV Cluj-Napoca and other botanical gardens, research centers and different institutions.
From the 46 de genotypes 34 belong to C. officinalis species, five to C. arvensis species, two to C. alata, two to C. stellata, and two genotypes to C. suffruticosa and one genotype to C. tripterocarpa.
Table 1
Species, genotype and prevalence of the studied biological material No. Species Genotype Prevalence 1 C. officinalis 122GE2822-0002 Denmark Botanic Garden, Universitat of Copenhagen 2 C. officinalis 123GEHortus Hudae Denmark Botanic Garden, Universitat of Copenhagen 3 C. arvensis 121GE2822-0001 Denmark Botanic Garden, Universitat of Copenhagen 4 C. stellata Cav. 124GE2822-04 Denmark Botanic Garden, Universitat of Copenhagen 5 C. officinalis C. officinalis L.D.a Germany (Berlin), Humblod-Universität zu Berlin, Institut für Biologie 6 C. officinalis C. officinalis L.B Belgique (Gembloux), Faculté universitaire des sciences agronomiques 7 C. arvensis C. arvensis L.B Belgique (Gembloux), Faculté universitaire des sciences agronomiques 8 C. officinalis C. officinalis F.a France Ville de Rouen, Jardin Botanique 76100 Rouen 9 C. arvensis C. arvensis F France Ville de Rouen, Jardin Botanique 76100 Rouen 10 C. officinalis C. officinalis SLO Slovenia Hortus Botanicus Ljubljana 11 C. arvensis C. arvensis SLO Slovenia Hortus Botanicus Ljubljana 12 C. officinalis cv. Probinelifera Nr.215 Germany (Deutschland) Botanscher Garten der Universität, 3703 Göttingen 13 C. officinalis cv. Prolifera Nr.214 Germany (Deutschland) Botanscher Garten der Universität, 3703 Göttingen 14 C. officinalis Bon-Bon Orange Latvia Seed Exchange, National Botanic Garten Salasplis, LV-2169 15 C. officinalis Bon Bon Mix’ Ukraine Hortus Botanicus Fominianus, Kiev 16 C. officinalis C. officinalis UK Ukraine Hortus Botanicus Fominianus, Kiev 17 C. officinalis C. officinalis L.D.b Germany Botanischer Garten, Universität Ulm D-89069 18 C. officinalis C. officinalis L.F.b France Botaniquest et Zoologiques, Arboretum National de Chevreloup 19 C. officinalis C. officinalis L.D.c Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 20 C. officinalis cv. Pacific-Riesen Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 21 C. officinalis cv. Radio Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 22 C. alata cv. Rech.f. Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 23 C. arvensis L. C. arvensis L. Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 24 C. stellata C. stellata Cav. Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 25 C. suffruticosa C. suffruticosa Vahl. Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 26 C. tripterocarpa C.tripterocarpaRupr. Germany Universitat Bayreuth Ökolog-Botanischer Garten D-95440 27 C. officinalis C. officinalis L.F.c France Jardin Botanique, 44094 Nantes cedex 1 28 C. officinalis C. officinalis L.D.d Germany Botanischer Garten J.W. Goethe-Universität D-60054 29 C. officinalis C. officinalis L.D.e Germany Botanischer Garten, Martin-Luther-Universität D-06108 30 C. officinalis C. officinalis L.PL Poland Lublin, Hortus Farmacognosticus Academ. Med. Ul.W.Chodźki120-093 31 C. officinalis C. officinalis L.D.f Germany (Chemnitz), Botanischer Garten, Grünflächenamt 32 C. officinalis C. officinalis D.g Germany (Deutschland), Bot. Garten der Cristian-AlberchtsUniv. Kiel, D-24098 33 C. officinalis C. officinalis I Italy (Urbino) Instituto e Orto Botanico Universitat di Urbino, 61029 34 C. officinalis C. officinalis D.h Germany Botanischer Garten der Cristian-Alberchts-Universität Kiel 35 C. officinalis cv. Prycosnovjenie Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 36 C. officinalis cv. Pacific Beauty Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 37 C. officinalis cv. Gaicha Gril Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 38 C. officinalis cv. Fiesta Hitana Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 39 C. officinalis cv. Zelenoye Serdtse Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 40 C. officinalis cv. Rozovyi Sjurpriz Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 41 C. alata C. alata UK Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 42 C. suffruticosa C. suffruticosa Ukraine National Botanical Garden, Timirjazevska, 1, Kyiv, 01014 43 C. officinalis C. officinalis A Austria Botanischer Garten Landesregierung Klagenfurt A-902 44 C. officinalis cv. Pacific Czech Republic Masarykova Univerzita Brne, Lékařská faculta 66243 Brno 45 C. officinalis cv. Plamen Czech Republic Masarykova Univerzita Brne, Lékařská faculta 66243 Brno 46 C. officinalis C. officinalis AZ Azerbaijan Republic Central Botanical Garden, Badamdar, AZ 1073
Experimental plot layout During the three years of experiment (2008, 2009, 2010) the experimental plots where
emplaced in Agrobotanical Garden of USAMV Cluj-Napoca. In 2008 the culture was established in household sector (approx. 316 m2), in 2009 in the biodiversity sector, sub-experimental
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agricultural habitats (about 81.45 m2) and in 2010, again in the biodiversity sector, sub-agricultural habitats experimental, represented by another parcel (63.00 m2).
Appling of Griffing model II dialel breading method Dialel breading methods, introduced by Griffing permit the estimation of CGC and CSC
effects (GRIFFING, 1956). The breading intraspecific scheme is presented in Table 2:
Table 2. Intraspecific breading combinations enrolled and breading scheme (dialel II Griffing)
♀ ♂ cv.Prycosnovjenie cv.Pacific Beauty Bon Bon Mix’ Bon-Bon Orange cv.Prycosnovjenie Yes Yes Yes Yes cv.Pacific Beauty - Yes Yes Yes Bon Bon Mix’ - - Yes Yes Bon-Bon Orange - - - Yes
Study of main seed characteristics at 46 Calendula genotypes The biological material represented by 46 genotypes belonging to six Calendula species
(C. arvensis, C. alata, C. officinalis, C. stellata, C. suffruticosa, C. tripterocarpa) has been analyzed from seed main characteristics. Determinations where established in laboratory conditions at the Biochemistry Department, Faculty of Agriculture, USAMV Cluj-Napoca. The studied characters where: length, width, thickness, weight, shape, through standard notes (1=cane shape - 5=circular shape), seed color, through standard notes (1=cream - 5=dark gray) and tegument aspect (1=smooth rough - 5= strongly serrated).
Study of main plants characteristics Enrolled measurements: height, 10 cm above soil diameter, no. of branches, insertion
angle, no. of leaf, leaf length and width; number of buds, flowers and fruits per plant, corolla and disc diameter; tubular and ligulate flower color, through standard notes (1=yellow - 7=dark brown), number, length and petal width.
Plant behaviour to pest and diseases attack During the experimental years was established the behavior of marigold plants at pest and
diseases attack, the obtained data where average calculated for each genotype. Attack frequency (F%), was determined as relative value for the total number of plants (n), reported at the number of plants or vegetative organs observed (N): F%= n x 100/N
The qualitative expression of intensity attack was calculated with the mathematical formula: I% = ∑(i x f)/100
i = grade or % covering percent (class); f = the number of attack cases related to each grade or class; n = number of attack cases.
Degree of attack: G.a.%= F% x I%/100
Methods used for the determination of the philogenetic relationships
Molecular markers method: the development of molecular biology techniques based on DNA markers led to enlargement for determining new plant genotypes, assuring a better understanding of relationships between species, gender and family.
PCR discovery by MULLIS, in 1984 (FORE and all., 2006) represented a key role in molecular genetics (RUSU, 2007). The procedure use two specific primers based upon the polymorphism of parental forms at allelic level, regarding the length of the amplification product (POREBSKI and all., 1997). Regarding the length of the primers can be established three different variants: RAPD technique (Randomly Amplified Polymorphic DNA), (WILLIAMS
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and all., 1990; PARENT and PAGE, 1992; GRAHAM and MCNICOL, 1994), DAF technique (DNA Amplification Fingerprinting), and AP-PCR technique (Arbitrary Primers PCR).
The vegetative material: represented by young leafs from 45 genotypes belonging to six Calendula species was immersed in liquid nitrogen. DNA isolation was established in the Genomic Laboratory from the Vegetal Biotechnology Department of USAMV-CN in 2009 using the extraction protocol of LODHI and all. (1994), improved by POP and all. (2004).
RAPD Random Amplified Polymorphic Method: this protocol was described for the very first time by WILLIAMS and all., (1990) and is based upon the polymorphism of parental forms at allelic level. Generally speaking each primer determines the sequence amplification from various origins of the genome, this protocol is being considered to be the highest efficient way of investigating DNA polymorphism between individuals (TINGEY and DEL TUFO, 1993).
Artificial breading system
Intraspecific and interspecific breading: the intarspecific breading takes apart between individuals belonging to the same species, not being able to raise fertility issues to the obtain hybrids. Problems can occur only in cases of gametophyte or sporofitic incompatibility or due to different cases of parental sterility.
The interspecific breading involves the breading of individuals belonging to different species, related to the same gender. During the experimental years was use dialel breading, which involves the breading of parental forms belonging to a group with all the others genitors.
Artificial breading protocol: The artificial breading was executed by partial protocol
stages, regarding the lack of bibliography for the induction of artificial breading at Calendula. The general protocol of artificial breading was adopted as describe in different breading treaty ARDELEAN (1986); ARDELEAN and all. (2006); SAVATTI and all. (2004). Where accomplishes intraspecific and interspecific breadings at: C. arvensis, C. alata, C. stellata, C. suffruticosa, free pollination and auto pollinations.
EXPERIMENTAL DATA PROCESSING
Statistically analysis of variance: the experimental data where statistically related, using ANOVA test for the main seed and plant characteristics at 45 Calendula genotypes. The obtained results were presented as average per experimental variant included in synthesis tables (ARDELAEAN and all., 2006).
Genetically analysis of variance: the mathematical model for CGC and CSC analysis is presented in Table 3 (GRIFFING, 1956 a; GRIFFING, 1956 b).
Heritability coefficient calculation: where generated using consecrated formulas, in relation with cited literature (PIEPHO and MÖHRING, 2007).
- Heritability coefficient broadly: H2 = 22
2
2
2
EG
G
P
G
sss
ss
+=
- Heritability coefficient restrained: h2 = 22
2
2
2
EG
AG
P
G
sss
ss
A
+=
6
Table 3. Genetically analysis of variance for experimental method II
Expected means squares Source df SS MS Model I
General combining ability p-1 SSg MSg σ2 + (p+2)/(p-1)∑gi2
Specific combining ability p(p-1)/2 SSg MSs σ2 + 2∑i∑jsij2/[p(p-1)]
Error Sum(ni)-dfg-dfs SSe MSe σ2 CGC = general combining ability; df = degrees of freedom; CSC = specific combining ability; SSG = ∑i(xi. + x.i)2/(2(p-2)) – 2x..
2/(p(p-2); CPG = components of genetic parameters; SSS = ∑i, i≤j∑(xij+xji)2/2 – ∑i(xi. + x.i)2/(2(p-2))+ x..
2/[(p-1)(p-2)]; SS = sum of squares error; SSR = ∑i, i≤j∑(xij-xji)2/2; MS = mean of squares error; SSE = SSE(Anova)/n
Statistical and mathematical methods used for calculation and interpretation of
molecular variability study: the similitude index (S) was calculated, genetic distances and finally the study was concluded by the realization genotypes dendogram. The presence or the miss presence of genotype fragments was notated with the mark 1 or 0. The results where introduced in a excel spreadsheet calculating the Jaccard (Jij) coefficient: Jij = Cij/(ni +nj-Cij), where: Cij = no. of identical lanes; ni, nj = total lanes no. at genotype i and j. The dendogram was accomplished using genetic distances calculated with the Jaccard (Jij) coefficient (SAITOU and NEI, 1987; WEISING and all., 1995). For the dendogram construction was used Neighbor-Joining method: Free Tree software, preview of the dendogram with Tree View software and the philogenetic tree was translated using SAITOU and NEI (1987) methods.
RESULTS AND DISCUSIONS
Results regarding the variability of main seed characteristics at 46 Calendula genotypes Calendula plants depending the species and genotype form different types achene’s have
an increased polymorphism (SHISHIN, 1962; ACOSTA and all., 2001). The inner achene’s seeds are smaller with circular shape, the exterior placed seeds are larger and less circular (ISMAGILOV and CARJA, 2000; GONCEARIUC, 2001).
The study of main seeds characteristics has its importance for identifying different Calendula genotypes, and observing similitudes between seed main characteristics and their germination capacity. The average values of length, width (mm) weight (mg), thickness (mm) of seeds and plant emergence capacity at 46 Calendula genotypes are presented in Table 4.
The seeds length recorded different values, with a variance amplitude between 8.00 mm at C. arvensis F and 25.67 mm at C. alata UK.The seed width recorded values between 1.34 mm (C. arvensis F.) and 4.07 mm (cv. Rech. f.), with a general average of the experiment situated at 2.59 mm. The seeds weight recorded an ample variation between genotypes with set limits between 5.32 mg at C. arvensis F, and 21.06 mg at C. alata UK.
The smallest recorded seed weight was at C. arvensis F, C. arvensis SLO, Bon Bon Mix’, C. officinalis L.F.c, C. officinalis I, cv. Prycosnovjenie, cv. Fiesta Hitana, cv. Rozovyi Sjurpriz, cv. Pacific, cv. Plamen, C. officinalis AZ, with significant negative deviations compared with the experiment average (12.27 mg), considered as experimental control. The genotypes that recorded the highest plant emergence percent where: C. officinalis L.BR with 78.3%, C. officinalis L.B. with 78.0% and C. officinalis L.Fa., C. officinalis L.Fb. with 76.7%, all with superior differences, statistically assured by compare with the experiment average. The variability coefficients for main seeds characteristics recorded values situated between 32.5% (for seed length), 38.0% (for seed width), 26.6% (for seed thickness) and 42.6% (for seed weight).
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Table 4. Length, width, thickness, weight and plant emergence capacity at Calendula
Seeds length Seeds width Seeds thickness Seeds weight Plant emergence No.
of entry
Genotype (mm) (s%) (mm) (s%) (mm) (s%) (mg) (s%) (%) (±d)
1 122GE2822-0002 10.53ooo 22.7 2.37- 34.8 2.84- 29.2 12.02- 29.2 56.7 - 2 123GEHortus Hudae 18.47- 36.8 2.38- 34.3 3.33(*) 44.2 17.36*** 32.0 50.0 - 3 121GE2822-0001 18.60*(*) 25.0 2.32- 31.6 3.05- 67.5 18.21** 46.9 68.6 - 4 124GE2822-04 21.20*** 22.8 1.94- 34.7 2.16- 43.5 12.87- 41.3 51.4 - 5 C. officinalis L.BR 14.87- 27.7 2.40- 21.8 3.80*(*) 49.4 19.05*** 24.1 78.3 ** 6 C. officinalis L.B 10.20ooo 28.3 1.75ooo 20.8 2.21oo 21.4 11.27- 26.4 78.0 ** 7 C. arvensis L.B 16.13- 32.1 2.28- 43.0 1.93ooo 29.0 12.48- 63.9 0.0 ooo 8 C. officinalis F.a 10.80ooo 23.5 2.35* 18.0 3.58** 41.3 14.06 20.3 76.7 ** 9 C. arvensis F 8.00ooo 28.7 1.39ooo 11.3 1.34ooo 17.1 5.32ooo 27.3 33.8 -
10 C. officinalis SLO 12.73o 36.4 1.80o 30.5 2.22- 43.2 10.85- 27.2 63.3 - 11 C. arvensis SLO 11.87o 51.6 2.75** 34.8 3.16- 50.0 9.17oo 46.1 65.0 - 12 cv. Prolifera Nr.215 16.27- 39.1 2.24- 12.0 2.59- 12.0 11.61- 41.9 56.7 - 13 cv. Prolifera Nr.214 11.93oo 41.4 2.45- 31.3 2.38- 28.3 12.74- 31.7 61.7 - 14 Bon-Bon Orange 18.93* 32.3 2.31* 17.0 2.82- 47.1 18.21*** 33.9 68.3 - 15 Bon Bon Mix’ 10.60ooo 28.0 2.10- 28.1 2.01ooo 21.3 7.69ooo 31.4 51.8 - 16 C. officinalis UK 20.67*** 11.4 2.45(*) 24.0 2.51- 34.2 16.83** 33.8 61.1 - 17 C. officinalis L.D.a 16.53- 35.6 1.82 o 26.4 2.65- 30.6 11.69- 50.3 31.7 - 18 C. officinalis L.F.b 18.93*** 15.3 2.45(*) 29.5 3.01- 29.3 16.21*** 25.4 76.7 ** 19 C. officinalis L.D.b 15.47- 40.1 2.05- 30.6 2.83- 51.2 12.11- 48.6 68.0 - 20 cv. Pacific-Riesen 15.60- 39.1 2.45** 16.2 2.51- 25.0 14.59* 30.6 68.5 - 21 cv. Radio 19.40(*) 39.7 2.65*** 22.9 3.53* 47.2 16.32- 58.0 63.3 - 22 cv. Rech.f. 19.87* 41.2 2.99** 48.1 4.07** 52.4 17.31- 68.3 66.7 - 23 C. arvensis L.D 12.93oo 29.9 1.78- 45.7 2.31- 55.0 9.79- 54.2 70.0 * 24 C. stellata Cav. 16.00- 32.9 2.23- 29.4 2.78- 45.9 11.27- 54.7 53.3 - 25 C. suffruticosa Vahl. 15.80- 40.4 1.88- 41.4 2.75- 86.8 10.76- 58.3 60.0 - 26 C.tripterocarpaRupr 14.60- 29.2 2.87* 51.1 3.66- 47.2 11.04- 57.1 53.3 - 27 C. officinalis L.F.c 12.67oo(o) 21.0 1.84ooo 11.6 2.32(o) 22.5 10.20oo(o) 21.9 48.3 - 28 C. officinalis L.D.c 15.20- 39.2 1.87- 27.8 2.73- 54.1 13.25- 45.4 48.3 - 29 C. officinalis L.D.d 15.27- 38.5 1.79oo 23.0 2.79- 54.0 11.60- 41.6 41.7 - 30 C. officinalis L.PL 13.93- 37.4 1.79o 29.7 2.02ooo 31.2 8.74o 74.2 33.3 - 31 C. officinalis L.D.e 19.87** 30.6 2.44- 33.2 3.01- 60.7 16.83 46.1 48.3 - 32 C. officinalis D.f 16.20- 30.7 1.82- 47.4 2.55- 56.7 10.93- 33.2 50.0 - 33 C. officinalis I 14.33- 53.8 1.51ooo 29.0 2.13(o) 44.6 8.69oo 54.4 37.8 34 C. officinalis D.g 14.80- 41.0 1.99- 30.5 2.96- 63.9 10.84- 59.8 18.7 oo 35 cv. Prycosnovjenie 12.00oo 36.1 1.70ooo 14.4 2.21- 37.5 7.15ooo 65.7 17.1 oo 36 cv. Pacific Beauty 16.33- 44.6 1.63ooo 21.8 2.46- 33.0 10.61- 46.3 40.0 - 37 cv. Gaicha Gril 14.13- 40.7 1.77ooo 22.5 1.90ooo 17.5 9.79- 53.2 26.7 o 38 cv. Fiesta Hitana 15.47- 36.4 1.92- 19.6 2.01ooo 20.4 9.41ooo 30.6 46.7 - 39 cv. Zelenoye Serdtse 17.33- 24.2 1.86ooo 11.6 2.59- 39.0 9.93- 55.8 23.3 o 40 cv. Rozovyi Sjurpriz 13.67- 35.1 1.95o 11.5 2.19ooo 16.4 8.59ooo 42.7 23.3 o 41 C. alata UK 25.67*** 9.4 3.34*** 21.6 2.97(*) 24.9 21.06*** 34.0 31.1 - 42 C. suffruticosa 22.00*** 17.3 2.30- 25.2 2.70- 23.7 19.12*** 32.5 33.3 - 43 C. officinalis A 14.47- 33.9 1.75ooo 15.4 1.89ooo 24.9 10.18- 46.0 53.3 - 44 cv. Pacific 16.40- 37.1 1.63ooo 18.2 1.89ooo 29.4 9.20oo 39.2 36.7 - 45 cv. Plamen 14.60- 24.0 1.70ooo 17.5 1.66ooo 16.5 8.19ooo 42.4 40.0 - 46 C. officinalis AZ 14.60- 32.5 1.67ooo 21.5 2.29- 27.4 9.39ooo 29.8 30.0 - Experiment average (Mt.) 15.56 32.5 2.59 38.0 2.10 26.6 12.27 42.6 48.8 -
LSD 5% = 20.3 LSD 1% = 26.8
LSD 0.1% = 34.7 *, **, ***/o, oo, ooo Significance for P<0.05; 0.01 and 0.001 (*, **, *** positive; o, oo, ooo negative)
Results regarding the variability of main plant characteristics at 45 Calendula genotypes Compared with the seed main characteristics when where analyzed 46 Calendula
genotypes, for the main plant characteristics where observed only 45 genotypes because C. arvensis L.B (Belgium) didn’t emerge.
The average values of plant height, no. of ramifications, flowers/plant and number of petals/corolla are presented in Table 5.
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Table 5. Plant height, no. of main branches, no. of flowers and petals in corollaat 45 Calendula genotypes
Plant height No. of main branches No. of flowers Petals in corolla No. of
entry Genotype
(cm) (s%) Average (s%) Average (s%) Average (s%) 1 122GE 2822-0002 33.56ooo 28.5 11.41*** 23.0 12.18ooo 51.6 30.26ooo 13.4 2 123GE Hortus Hudae 37.60oo 19.0 9.33- 20.5 7.07ooo 45.2 35.40ooo 32.7 3 121GE2822-0001 55.00*** 16.5 10.37*** 22.9 20.60- 52.2 20.37ooo 18.4 4 124GE2822-04 39.15oo 28.5 4.85ooo 17.8 11.70ooo 27.7 21.63ooo 17.4 5 C. officinalis L.D.a 36.13ooo 31.4 16.38*** 41.0 7.43ooo 66.7 28.66ooo 43.7 6 C. officinalis L.B 31.06ooo 48.0 3.89ooo 69.9 4.06ooo 106.1 25.11ooo 66.4 7 C. officinalis F.a 36.19ooo 28.8 10.58*** 35.5 16.46- 51.0 38.17ooo 8.0 8 C. arvensis F 44.35- 17.0 4.59ooo 19.0 32.24*** 27.5 20.06ooo 13.9 9 C. officinalis SLO 42.11o 18.9 8.97- 20.8 16.50ooo 23.1 26.89ooo 7.6
10 C. arvensis SLO 67.72*** 13.8 7.36ooo 19.8 19.77- 28.7 15.87ooo 7.7 11 cv. Prolifera Nr.215 49.88- 34.2 5.59ooo 22.5 5.50ooo 51.9 42.85o(o) 42.4 12 cv. Prolifera Nr.214 59.65*** 35.2 13.43*** 33.8 11.00ooo 53.9 115.51*** 60.5 13 Bon-Bon Orange 52.12* 30.3 11.95*** 36.6 16.83ooo 36.5 41.66ooo 25.8 14 Bon Bon Mix’ 41.71- 38.2 6.79- 57.0 2.86ooo 71.1 97.64** 54.4 15 C. officinalis UK 30.64ooo 40.5 4.91*** 56.5 2.00ooo 86.6 19.91ooo 79.8 16 C. officinalis L.D.b 44.95- 37.7 10.00- 47.8 6.00ooo 78.6 26.47ooo 45.8 17 C. officinalis L.F.b 50.54- 34.2 10.50** 40.5 16.89o(o) 63.5 27.89ooo 32.6 18 C. officinalis L.D.c 43.29- 48.5 9.18- 60.7 14.53o(o) 77.0 24.44ooo 52.0 19 cv. Pacific-Riesen 41.43- 36.0 8.11- 38.9 10.24ooo 91.2 51.35- 105.5 20 cv. Radio 45.89- 31.0 7.89- 41.9 16.08oo 64.0 49.29- 31.6 21 cv. Rech.f. 61.15*** 29.6 9.45- 34.4 32.10*** 48.1 25.65ooo 4.9 22 C. arvensis L. 58.12*** 32.6 8.79- 36.6 73.07*** 33.4 24.83ooo 6.0 23 C. stellata Cav. 60.53*** 27.0 9.22- 33.9 83.03*** 32.6 25.00ooo 5.3 24 C. suffruticosa Vahl. 65.89*** 23.0 9.31- 29.4 98.00*** 26.7 25.06ooo 8.1 25 C. tripterocarpa Rupr. 84.06*** 12.6 11.53*** 19.2 96.53*** 19.0 24.66ooo 6.3 26 C. officinalis L.F.c 51.45* 29.7 9.14- 47.8 16.86o 61.2 33.00ooo 41.4 27 C. officinalis L.D.d 71.24*** 14.0 9.10- 31.3 17.24oo 33.8 51.69- 18.0 28 C. officinalis L.D.e 31.72ooo 32.0 7.44- 53.6 26.16- 60.2 22.88ooo 46.2 29 C. officinalis L.PL 43.85- 41.4 7.40- 55.7 13.85ooo 65.6 176.30*** 43.8 30 C. officinalis L.D.f 46.87- 35.6 9.52- 49.1 23.52- 65.5 31.52ooo 61.0 31 C. officinalis D.g 47.67- 38.5 7.50- 54.0 17.07o 52.8 31.90ooo 46.5 32 C. officinalis I 35.85oo 32.0 6.85- 50.5 8.69ooo 64.3 60.31- 33.6 33 C. officinalis D.h 29.00oo 49.5 5.50o 67.8 3.50ooo 109.5 25.33ooo 50.8 34 cv. Prycosnovjenie 38.88- 27.2 10.75* 24.2 13.75o 63.1 14.00ooo 62.6 35 cv. Pacific Beauty 40.00- 46.9 7.58- 56.6 10.75ooo 56.5 82.08* 58.7 36 cv. Gaicha Gril 43.16- 34.7 9.19- 44.3 12.61ooo 52.2 194.00*** 41.5 37 cv. Fiesta Hitana 28.79ooo 34.5 6.29o 62.8 3.43ooo 107.1 159.57** 78.7 38 cv. Zelenoye Serdtse 31.57o 53.2 7.29- 48.7 3.00ooo 101.8 181.86(*) 97.0 39 cv. Rozovyi Sjurpriz 22.00ooo 61.7 5.71- 83.8 3.14ooo 90.8 107.00(*) 68.8 40 C. alata UK 62.40* 17.1 9.80- 19.6 54.20**(*) 40.4 23.20ooo 3.6 41 C. suffruticosa 60.60- 27.3 8.60- 39.9 58.00*** 36.2 22.80ooo 3.7 42 C. officinalis A 29.44ooo 34.8 8.69- 62.9 18.19- 55.5 24.25ooo 50.2 43 cv. Pacific 55.91* 24.9 8.00- 31.6 10.82ooo 58.7 60.45- 36.7 44 cv. Plamen 26.00ooo 66.7 5.50ooo 40.7 2.17ooo 103.8 126.75** 88.6 45 C. officinalis AZ 44.11- 34.2 9.89- 49.4 15.89- 69.0 29.89ooo 19.5
Experiment average 45.63 32.8 8.54 41.2 21.46 59.2 52.08 38.7 *, **, ***/o, oo, ooo Significance for P<0.05; 0.01 and 0.001 (*, **, *** positive; o, oo, ooo negative)
Compared with the experiment average, statistically assured values regarding the number
of branches per plant were observed at 17 genotypes. The no. of flowers/plant is considered one of the most important characters for Calendula
ornamental capacity. The variability of petals/corolla records high values, with limits between 14.00 (cv. Prycosnovjenie) and 194.00 (cv. Gaicha Gril).
Phenotypic correlation results for main seed and plant characteristics at Calendula During the experimental years where observed different characteristics, related with
phenotypic correlations, statistically assured, positive or negative (Table 6.). Positive phenotypic correlations, very significant where observed between: seed weight and seed germination; seed
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weight and no. of branches; plant height and no. of branches; plant height and no. of flowers/plant. During specific observation was confirmed that the more weight recorded at seeds, their germination was better (BACIU and SESTRAŞ, 2008).
Once with the seed weight increase, proportionally increased the no. of ramifications/plant. On the other hand the no. of petals was negatively related with weight and seed germination, plant height, no. of flowers/plant.
Table 6.
Phenotypic correlations between main seed and plant characteristics at Calendula
Related characteristics Seed germination Plant height No. of branches
No. of flowers
No. of petals
Seed weight 0.420*** 0.226* 0.433*** 0.095- -0.337oo(o) Seed germination 0.220* 0.294** 0.115- -0.362ooo Plant height 0.377*** 0.689*** - 0.282oo No. of branches 0.234* - 0.156- No. of flowers - 0.325oo
r 5% = 0.205; r 1% = 0.267; r 0.1% = 0.338
Results regarding the behavior of 45 Calendula genotypes at aphid attack The species Aphis fabae (aphids) harm by direct attack and secondary damage through
the secretion of "honey dew" considered the main vectors of virus diseases found in plants (DIXON, 1998; OLTEAN and all., 2004).
Aphids are common pests (specific for) cultures of Calendula with a presence in culture, very important as terms of intensity and frequency of its attacks (BACIU and all., 2009).
From the 45 genotypes of Calendula studied in 2008, attack of Aphis fabae was observed at 19 genotypes, 26 of them presented lack of attack. Throughout genotypes attacked by Aphis fabae where observed differences of frequency and intensity (Table 7.).
Table 7.
Frequency (F%), Intensity (I%) and Degree attack (G.a.%)
Frequency (F%) Intensity (I%) Degree attack (G.a.%) No. variant Variant/Genotype (F%) Significance (I%) Significance (G.a.%) Significance 1 122GE 2822-0002 2.94 - 30.00 *** 0.88 - 2 123GE Hortus Hudae 26.60 *** 21.25 ** 5.65 * 3 121GE2822-0001 17.10 ** 34.17 *** 5.84 *(*) 4 124GE2822-04 14.81 * 25.50 *** 3.78 - 9 C. officinalis 7.89 - 31.67 *** 2.43 - 10 C. arvensis 12.82 - 25.00 *** 3.20 - 11 cv. Prolifera Nr. 215 14.70 * 21.00 ** 3.08 - 18 C. officinalis L. 14.70 * 25.00 *** 3.67 - 19 cv. Pacific-Riesen 10.81 - 13.75 - 1.49 - 21 cv. Rech.f. 7.50 - 30.00 *** 2.25 - 22 C. arvensis L. 14.28 - 26.67 *** 3.81 - 23 C. stellata Cav. 12.50 - 20.00 ** 2.50 - 26 C. officinalis L. 10.34 - 18.33 * 1.89 - 28 C. officinalis L. 24.00 *** 17.50 * 4.20 - 30 C. officinalis L. 22.58 *** 15.00 - 3.39 - 40 C. alata 20.00 ** 15.00 - 3.39 - 41 C. suffruticosa 40.00 *** 20.00 ** 8.00 *** 42 C. officinalis 25.00 *** 15.00 - 3.75 - 44 cv. Plamen 25.00 *** 16.67 (*) 4.17 -
Average of the experiment (Mt.) 17.03 - 22.18 - 3.54 - *, **, ***/o, oo, ooo Significance for P<0.05; 0.01 and 0.001 (*, **, *** positive; o, oo, ooo negative)
The highest frequency of attacks with an average value (F = 40.0%) was observed at C.
suffruticosa, prevailed from the National Botanic Garden of Ukraine, Kyiv, followed by genotype C. officinalis genotype 123GE prevailed from the National Botanical Garden Denmark,
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Copenhagen (F% = 26.6). For F% attack, 11 genotypes recorded significant statistical differences and 15 for I% attack and only three genotypes recorded statistical differences for Ga%. Table 8. presents estimated attacks regarding the number and percentage of genotypes enrolled in 6 categories (disregarding 57.8% genotypes with no attack degree).
The highest value recorded for the frequency of attack (22.2%) was related to the medium category of attack, while the same proportion to the intensity of the attack was recorded in the category of very strong attacks. Regarding the degree of attack, the largest proportion of genotypes was assigned to the category of poor attack (33.3%).
Table 8.
Number and percentage of genotypes included in different categories of attack, related to Frequency, Intensity and Attack Degree
Frequency (F%) Intensity (I%) Attack Degree (G.a.%) Attack estimation (F%, I%, G.a.%) No. % No. % No. %
Without attack (Attack % = 0) 26 57.8 26 57.8 26 57.8 Very weak attack (Attack % = 0.1-1) - - - - 1 2.2 Weak attack (Attack % =1.1-5.0) 1 2.2 - - 15 33.3 Medium attack (Attack % = 5.1-15) 10 22.2 4 8.9 3 6.7 Strong attack (Attack % = 15.1-20) 2 4.5 5 11.1 - - Very powerful attack (Attack % > 20.1) 6 13.3 10 22.2 - -
Total genotypes 45 100.0 45 100.0 45 100.0
Results regarding the philogenetic relationships between different species and genotypes Using molecular markers represented a strong growing effectiveness for the breading
procesees in both plants and animals regnum, their applicability is demonstratedm in: identification and genetic taxonomy, development of genetic maps, phylogeny studies (VOS et al., 1995). The research goals where focused upon the analises of the diversity of six species of Calendula with origin from 12 countries at the molecular level by DNA analysis and phylogenetic tree preparation.
DNA isolation results: the average amount of DNA obtained was 297.09 ng/ml. Results of DNA amplification: profile bands formed by-products of amplification was monitored by gel electrophoresis RAPD technique, often used for this purpose (BASSAM et al., 1991). 15 primers were tested, whose high-guanine and cytosine ranged between 70% -80% in all 45 samples. Of the 15 primers used for amplification decameri, only five were amplified (33.33%), they were selected for RAPD analysis.
Although it is considered that each DNA fragment is a locus (Landry et al., 1994), however, it’s posible that some bands may represent multiple locus, which have migrated together (PAMFIL et al., 2000). JÄNTSCHI et al. (2009) suggests in this regard to elucidate the genetic algorithms using nucleotide sequence analysis. By using the five primers produced 56 bands were polymorphic, achieving an average of 11.2 bands/primer.
Primer OPH-20 formed a no. of 11 polymorphic bands per sample. Amplification occurred at almost the same level for all samples, and generated polymorphisms by primer OPH-20 was very closed to the primer OPC-15. The highest number of bands was recorded at C. officinalis LDF genotypes, cv. Pacific Beauty, cv. Rozovyi Sjurpriz, Dg C. officinalis and C. stellata Cav. (Figure 1).
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Legend 1 - C. officinalis - cv. Pacific 2 - C. officinalis - Bon Bon Mix’ 3 - C. officinalis L.D.c 4 - C. officinalis L.D.e 5 - C. officinalis L.F.c 6 - C. officinalis - cv. Pacific-Riesen 7 - C. tripterocarpa - C. tripterocarpa Rupr. 8 - C. arvensis - 121GE2822-0001 9 - C. officinalis L.D.d 10 - C. officinalis AZ 11 - C. officinalis SLO 12 - C. officinalis L.PL 13 - C. stellata Cav. - 124GE2822-04 14 - C. officinalis D.h 15 - C. officinalis L.D.a 16 - C. officinalis - Bon-Bon Orange 17 - C. officinalis L.D.f 18 - C. officinalis L.B 19 - C. officinalis L.F.b 20 - C. arvensis L. 21 - C. alata UK 22 - C. arvensis F 23 - C. officinalis - cv. Pacific Beauty 24 - C. officinalis – cv. Rozovyi Sjurpriz 25 - C. officinalis - cv. Zelenoye Serdtse 26 - C. officinalis A 27 - C. officinalis - cv. Prolifera Nr.214 28 - C. suffruticosa - C. suffruticosa Vahl. 29 - C. stellata - C. stellata Cav. 30 - C. arvensis SLO 31 - C. alata - cv. Rech.f. 32 - C. officinalis -123GEHortus Hudae 33 - C. officinalis - cv. Fiesta Hitana 34 - C. officinalis - cv. Plamen 35 - C. officinalis - 122GE2822-0002 36 - C. officinalis D.g 37 - cv. Prolifera Nr.215 38 - C. officinalis I 39 - C. officinalis UK 40 - C. officinalis L.D.b 41 - C. officinalis F.a 42 - C. officinalis - cv. Radio 43 - C. officinalis - cv. Prycosnovjenie 44 - C. officinalis - cv. Gaicha Gril 45 - C. suffruticosa L - marker ADN 100 Kb
Figura 1. Reaction products amplification with OPAL-20 primer at Calendula OPA-18 primer generated a maximum of 10 bands/sample, with reduced polymorphism.
OPC-15 primer generated a maximum of 12 bands/sample with a very low polymorphism. Almost all the bands generated by primer OPC-15, amplified at the same level, except for genotypes: C. stellata Cav.124GE, cv. Gaicha Grill and C. officinalis 122GE.
OPAB-18 primer generated a maximum of 9 bands/sample, polymorphism generated by this primer is quite enlightening. Genotypes at Bon Bon Mix’, C. arvensis 121GE, C. officinalis AZ, C. stellata and C. hispida Cav.124GE UK, primer generated only one polymorphic band and the genotype of C. officinalis D.h, didn’t generated no band.
Dendogram result regarding the 45 genotypes belonging to six species of Calendula Using RAPD technique, the resulting dendogram, following tests carried out and data
processing was considered a true representation of the philogenetic relationships between different genotypes of Calendula and existing genetic diversity (Figure 2.).
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Figure 2. Dendogram of 45 Calendula genotypes belonging to six species
(Software: FREE TREE→TREE VIEW) In the generated dendrogram, genotypes from where genetic distances were small and
were grouped close, while genotypes from the higher genetic distances were placed further apart. In the dendrogram were used 45 genotypes belonging to six species of the Calendula gender: C. arvensis, C. hispida, C. officinalis, C. stellata, C. suffruticosa, C. tripterocarpa with origin from 12 countries. Regarding the dendrogram results can be observed a diversification of genotypes divided into four large groups separate, numbered for ease of interpretation with the letters from „A” to „D”.
The group noted with letter „A” included four subgroups that meet three species (C. arvensis, C. hispida and C. officinalis L.) considered the group that unites the greatest number of genotypes of this dendogram (17 genotypes). The group brings together two different phenotypically species, but because they possess the same active principles are used as medicinal herbs (C. officinalis L. and C. arvensis L.). They are only used as medicinal plants, due to their high content of active principles (BRÂNZILĂ, 2005; VĂTAVU, 2010).
The group noted with letter „B” unites five subgroups and 12 genotypes belonging to four species: C. officinalis (cv. Orange Bon Bon, cv. Prycosnovjenie, cv. Gaicha Grill, C. officinalis D.h. and C. officinalis L.D.a, C. officinalis L.F.b, LFB C. officinalis and C. officinalis LA, C. officinalis L.D.f), C. arvensis F., C. stellata Cav and C. suffruticosa.
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The group noted with letter „C” is divided into two subgroups and unites six genotypes belonging to two species (C. officinalis L. and C. suffruticosa Valh.).
The last group, entytled by „D” letter includes ten genotypes belonging to five species of the genus Calendula: C. officinalis, C. arvensis, C. hispida, C. stellata, C. tripterocarpa.
Obtained results during studies conducted at F1 Calendula hybrids prevailed through intraspecific dialel breadings, Griffing II model
Growth elements analyses at F1 hybrids: at Calendula, elements that determine the
growth and stems branching directly influence both decorative plant capacity and technological seeding distances, per unit area.
Hibrid height: experimental data regarding plant height at 10 Calendula F1 hybrid combinations obtained in a Griffing dialel type II scheme are represented in Table 9.
Within genotypes the variability of plant height limit was between 18.03% for the combination of cv. Pacific Beauty x Bon-Bon Orange and 53.61%, for the hybrid combination of cv. Prycosnovjenie x Bon-Bon Orange.
Table 9. Results regarding the height of F1 Calendula hybrids obtained from hybrid
combinations of the experiment
No. var.
Hybrid combination ♀/♂
Average height and average eror (cm)
Difference upon average (cm)
“t” value
Dif. siggnificance CV%
1 cv. Prycosnovjenie x cv. Prycosnovjenie 54.32±4.29 5.56 1.23 - 34,44 2 cv. Prycosnovjenie x cv. Pacific Beauty 52.30±3.19 3.55 1.02 - 29,27 3 cv. Prycosnovjenie x Bon Bon Mix’ 56.78±4.05 8.02 1.88 (*) 21,40 4 cv. Prycosnovjenie x Bon-Bon Orange 37.36±6.04 -11.39 -1.84 (o) 53,61 5 cv. Pacific Beauty x cv. Pacific Beauty 47.50±4.81 -1.26 -0.25 - 42,98 6 cv. Pacific Beauty x Bon Bon Mix’ 52.92±4.29 4.16 0.92 - 28,07 7 cv. Pacific Beauty x Bon-Bon Orange 40.00±4.16 -8.76 -2.00 o 18,03 8 Bon Bon Mix’ x Bon Bon Mix’ 41.31±2.01 -7.45 -3.07 oo 19,44 9 Bon Bon Mix’ x Bon-Bon Orange 50.57±4.04 1.81 0.43 - 42,29 10 Bon-Bon Orange x Bon-Bon Orange 46.19±3.12 -2.57 -0.75 - 34,39
Average of the experiment (Mt.) 48.76±1.01 - - - 35.95 *, **, ***/ o, oo, ooo Significance for P<0.05; 0.01 and 0.001 (*, **, *** positive; o, oo, ooo negative)
The obtained results after the genetical analyses for F1 hybrids regarding CMC and CSC
of genotypes are presented in Table 10. From the prevailed data we can observe that the parental forms used in the breading proceses for CSC effects are real, in the height growth in hybrid combinations, and not instead of CMC.
Table 10.
Analysis of variances for general and specific combining ability (GCA and SCA) of genotypes used as genitors for F1 progeny height
“F” value Variance source Sum of Squares (SP) Degrees of freedom
(GL) Variance (s2) “F” Calculated “F” Theoretical
CGC 103.75 3 34.58 1.89- 2.67; 3.91 CSC 287.82 6 47.97 2.63* 2.16; 2.92
Eroarea 2846.98 156 18.25 - - *, **, ***/ o, oo, ooo Significance for P<0.05; 0.01 and 0.001 (*, **, *** positive; o, oo, ooo negative)
Results synthesis for CGC and CSC effects of genotypes used as parental forms and CSC
constancy, are represented in Table 11. Following the results synthesis, the obtained data reveal that plant height was significantly influenced by the negative effects of the CGC, for cultivar Bon-Bon Orange.
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Table 11. The effects of SCA, GCA and SCA constancy on F1 Calendula hybrids height
of related on genotypes used as genitors
SCA Effect ♀/♂ Genitors cv. Prycosnovjenie cv. Pacific Beauty Bon Bon Mix’ Bon-Bon Orange
GCA Effect
SCA Constancy
cv. Prycosnovjenie 1.240- 1.705- 5.733- -9.919o 2.575- 58.726 cv. Pacific Beauty -0.623- 4.348- -4.806- 0.099- 13.528 Bon Bon Mix’ -7.701- 5.320- 0.544- 42.317 Bon-Bon Orange 4.703- -3.218o 76.827
LSD for GCA significance effect: DL 5% = 3.276; DL 1% = 4.318; DL 0,1% = 5.543 LSD for SCA significance effect: DL 5% = 8.459; DL 1% = 11.150; DL 0,1% = 14.311
Heritability of analized characteristics at F1 intraspecific Calendula hybrids, obtained by Griffin II model breading
Dialele breadings performed have estimated the effects of general combining ability and
specific combining ability of genotypes used as parental forms for manifestation of the characteristics analyzed in F1 hybrids.
Heritability in broad sense has different values, varying according to character (Figure 3.) the largest value consisting in the number of leaves on branches (H2 = 0.778).
0.419
0.343
0.4420.423
0.133 0.115
0.734
0.591
0.7780.749
0.4350.416
0.3250.287
0.2
0.4
0.6
0.8
Plant height Stemdiameter
Mainbranches
The angle ofinsertion
No. leaves The leaflength
The leafw idth
H h
Figure 3. Heritability coeficients regarded generaly and constrained for the main charachteristics of plant vigour at Calendula
All analyzed characters in F1 hybrids had experience under considerable genetic
determinism, is influenced more than 50% of genotype and a relatively lesser extent by environmental factors. The lowest rate of participation in the manifestation of the genotype data was obtained for the number of main branches (13.3%). The phenotypic expression of this character recording a significant importance for the environmental conditions, other H2
characters have values between 0.221 and 0.734. Even if the characteristics studied had experienced a remarkable genetic determinism, not all cases have additive.
This results from the analysis of the heritability values for the coefficients obtained in the narrow sense, the limits were very high, ranging between 0.115 (the no. of main branches) and 0.749 (no. of leaves/main branches). If for the number of leafs on main branches, with the largest value for h2, aditivity effects recorded manifestation of a considerable weight to characters such as the number of main branches, the length of the petals and corolla diameter (Figure 4.), no additivity and dominance effects were the major contributor, the epistatic effects play a key role.
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0.592 0.575
0.402
0.359
0.699 0.658
0.2930.275
0.3610.334
0.654
0.621
0.221 0.2
0.5060.487
0.2
0.4
0.6
0.8
No. flowersbuds No. flowers No. fruits Corolla diameter Disk diameter No. petals The petal lenght The petal width
H h
Figure 4. Heritability coeficients regarded generaly and constrained for the main
charachteristics of flowers and fruits at Calendula The no. of main branches is considered an important element for the decorative aspect
and plant vigor, as the value h2 character was low, indicating that the improvement of Calendula may confround difficulties for obtaining large numbers of genotypes with major ramifications through artificial breading, even that parental forms poseses this character.
CONCLUSIONS AND RECOMMENDATIONS
Due to multiple usages of plants many plant species belonging to Calendula gender especially Calendula officinalis L. enrich of the existing gene pool with new genotypes is imperative. For the selection of genitors as amounts necessary works to improve biological seed and vegetative material was analyzed phenotypic ally and molecularly. After conducting these tests and establishment of improvement targets, were selected maternal and paternal genitors for breading techniques that have been made.
Conclusions and recommendations resulted after pheonotypical analysis of 46 Calendula genotype seeds Both within the 46 genotypes studied, and the whole experience, the character with the
highest variability was represented by seed weight. Genotypes with the highest percentage of emergence where C. officinalis L.BR 78.3%, C. officinalis L.B with 78.0% AB, C. officinalis and C. officinalis LFa, LFb C. officinalis with 76.7%. All recorded differences were recorded and assured statistically than the experience average (48,8%), considered as experiment control.
Positive phenotypic correlations were recorded between certain characteristics of seed and plant emergence. Was observed that Calendula seeds with larger dimension, heavier, positively affects germination and plant emergence. The results confirm some data from the research literature, whereby between seed size and capacity of germination and plant emergence are closely linked positive (BRÂNZILĂ, 2007).
As potential genitors for seed weight and germination capacity, plant emergence we recommended the following genotypes: C. officinalis L.BR. and C. officinalis L.F.b.
Conclusions and recommendations resulted after fenotipical variability at 45 Calendula plants genotypes Research results assured that an ample variation between the 17 analyzed characteristics
allow a selection of genitors that fulfill the main research objects. For further breading processes
16
in obtaining plants with large height, the following genotypes must be considered: cv. Pacific, Bon-Bon Orange, cv. Prolifera Nr.124, C. officinalis L.D.d and C. officinalis L.F.c.
According to recorded data the highest number of flowers/plant was observed at C. tripterocarpa Rupr. and C. suffruticosa Vahl. Although these species have a large number of flowers/plant, the flowers are small with lack of decorative importance.
Regarding the correlations between the main seed and plant characteristics was observed that seed weight influence plant emergence was positively correlated with the phenotypic characteristics of ornamental plants, positive height and number of stems per plant, and negatively correlated with the number of petals/flower. Such correlations statistically assured can be used as selection index to improve the marigolds breeading processes.
Conclusions and recommendations regarding the behavior of 45 Calendula genotypes at aphid attack Aphis fabae From the 45 studied Calendula genotypes, 26 genotypes representing 42.2 % didn’t
presented attack degree. 19 genotypes representing 57.8% where attacked by aphids. Results obtained from the basis of determining F%, I% and G% of the aphids at 45 Calendula genotypes illustrates that even though 11 genotypes present statistical interpretable data and 15 different statistical interpretation attack, just three genotypes were obtained statistical differences Ga%.
Among the most sensitive genotype to attack of Aphis fabae, where observed species as C. suffruticosa (G% = 8%). Is important to mentioned that the response to the attack of aphids at Calendula plants manifested not only in different cultivars of C. officinalis also between different species. Different genotypes were found free from attack, which can lend itself as genitors in obtaining new cultivars, due to previous results (BACIU and SESTRAŞ, 2009) presented different decorative qualities.
Conclusions and recommendations regarding the variability through molecular markes methods (RAPD) Analysis was performed on six species of Calendula diversified at the molecular level by
DNA analysis and preparation of their phylogenetic tree. Results interpretation in terms of objectives pursued and interspcifice intraspecific hybridization in the genus Calendula, we can say that no special difficulties because of possible realization of sterility breading phenomenon.
However, such situations can occur when forms will be used as parental genotypes phylogenetically close to C. officinalis cv. Pacific Beauty and C. officinalis L.PL. that seem very related. The interspecific hybridization, genetic variability problem steep descent put less pressure because it is almost always a natural consequence of such crossings.
In this type of breading higher genetic distance between potential genitors or less gives an indication of the possible intensities intersterilitate manifestation of phenomenon and the viability of any possible hybrid seed produced.
Conclusions and recommendations regarding the results obtained in dialel and interspecific breading specific to Griffing II model After statistical data processing on plant height hybrids , made after the model of Griffing
dialela II, the hybrid combination Prycosnovjenie x Bon Bon Mix’ gave rise to progeny with the greatest vigor, with significant differences, compared with the average value experience of the observed character (48.76 cm).
At the parental forms used in experimental breadings the specific combining ability effects are real for determin F1 hybrids heights. However not those of the general combining ability. Interesting information regarding the effects of CMC and CSC, additivity phenomenon, dominance or epistazie upon transmission Calendula hybrid progeny were obtained and analyzed at other characteristics.
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Conclusions and recommendations regarding the results obtained after calculating heritability coefficients at intraspecific Calendula hybrids All the characteristics of intraspecific F1 analyzed hybrids had experience under
considerable genetic determinism influenced more than 50% of genotype and a relatively lesser extent by environmental factors. Heritability in the broad sense has different values, varying according to character, the highest value recorded for the number of leaves on the main branches (H2 = 0.778). The lowest rate of genotype participation in the manifestation data was obtained for the number of main branches (13.3%). The phenotypic expression of this character, outside the influence of genetic factors was influenced with a significant importance by environmental conditions. The other observed characters recorded values between 0.221 and 0.734 H2.
Number of main branches is an important element for the decorative aspect and plant vigor, as the value h2 character was low, revealing that the improvement of Calendula may occure difficulties in obtaining large numbers of genotypes with major ramifications through artificial hybridization, even if parental forms containe this character.
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